Skip to main content
eScholarship
Open Access Publications from the University of California

UC San Diego

UC San Diego Electronic Theses and Dissertations bannerUC San Diego

Insights into the functionality and targeting of PKA regulatory subunit RIalpha

Abstract

Regulatory subunits (R) of cAMP-dependent protein kinase (PKA) are differentially expressed and carry out various functions. Two general classes exist, RI and RII, as well as [alpha] and [Beta] isoforms within each class. Though the four isoforms share the same general domain organization, they are not functionally redundant. RI[alpha] is embryonically lethal in mice and cannot be compensated for by the other R isoforms. A change in RI[alpha] expression levels, higher or lower, is implicated in diseases, such as Carney complex (CNC). CNC is an autosomal dominant syndrome that is associated with RI[alpha] mutations, most of which are not expressed, resulting in a haploinsufficiency of RI[alpha]. I studied two mutations among the few that do get expressed, RI[alpha] R74C and RI[alpha](1-303). RI[alpha] R74C results in a mutation in the linker region, while RI[alpha](1-303) results in a truncation in the cAMP binding domain. My goals were to characterize these mutants in E. coli (Chapter 2) and in mammalian cells (Chapters 3 and 5) and to search for novel binding partners of RI[alpha] (Chapter 4). Purified RI[alpha](1- 303) showed a four-fold increase in PKA activation and RI[alpha] R74C dimers had a tendency to form disulfide- bonded tetramers. TAP-tagged constructs of RI[alpha] and RI[alpha] R74C were engineered and expressed in mammalian cells to make stable cell lines. Using diagonal gel electrophoresis, I showed that both constructs of TAP- tagged RI[alpha] formed heterodimers with endogenous RI[alpha]. No homodimers of endogenous RI[alpha] were observed when RI[alpha] R74C was expressed, which could explain the CNC phenotype. These stable cells were also used to study the RI[alpha] interactome. Using various affinity methods followed by mass spectrometry analysis, I discovered an A-kinase anchoring protein (AKAP), AKAP11, which binds with unusually high affinity to RI[alpha]. When RI[alpha] was overexpressed, it formed puncta, which were dependent on the disassociation from C-subunit and were reversible. In addition, RI-specific AKAPs abolished the puncta formation. Using indirect immunofluorescence and correlated light and electron microscopy, these puncta were found to localize to multivesicular bodies. In summary, I have identified an effect on the interchain disulfide bonding in RI[alpha], putative binding partners for RI[alpha], as well as a novel way of targeting RI[alpha]

Main Content
For improved accessibility of PDF content, download the file to your device.
Current View